The Use of X-rays To Determine the Mitotic and Intermitotic Time of Various Mouse Tissues* NORMANP. KNOWLTON,JR.,ANDWILLIAMR. Wi (Los Alamos Scientific Laboratory, Lo«Alamos, New Mexico) It has long been known that, as a general rule, only slight, if any, slowing of the mitotic process the sensitivity of various normal and malignant (4). The slope of the downward trend of the mitotic tissues to x-ray is proportional to the amount of index after irradiation must then be a measure of cell division which is occurring in the tissue. The the mitotic time. In other words, one would expect number of mitoses seen in a biopsy of a tumor is cells to be completing the mitotic process, but no one of the criteria for predicting the probable re cells would be entering middle prophase. Theo sponse of the tumor to irradiation (1). Since the retically, therefore, after a period equal to the amount of cell division, or more specifically the mitotic time, there would be no mitoses present mitotic index, is dependent upon the mitotic and which were later than the ones blocked at early intermitotic times (5), a study was undertaken to prophase. Actually, one would expect that a few determine which of these two factors is responsible cells would slip through the barrier at early pro- for differences in the mitotic index seen in various phase and also that a few cells which were past the tissues. barrier at the time of irradiation would be injured Many technics have been described by which the sufficiently to stop their progress through division mitotic time of various cells can be determined. (7)—the latter being manifested by various chro Hanging drop preparations (3, 8, 12) and tissue mosomal aberrations such as clumped metaphases, cui Iures (9, 13) have been used for direct observa chromosomal bridges of anaphase, etc. With the tion of cell division. The mitotic and intermitotic above theory in mind, a study was undertaken to times of some internal tissues have been estab determine the mitotic time of various mouse tis lished on the basis of the mitotic index and rate of sues by means of the fall in the mitotic index after growth of the tissue (2, 10). In the case of internal irradiation. organs in which there is no increase in size, this EXPERIMENTAL method is not applicable. If, however, it were pos CFy strain female mice, 8-16 weeks of age, sible to determine the mitotic time of these tissues which appeared healthy after 10 days' acclimation by some other means, then, knowing the mitotic at this laboratory, were used in these experiments. index, the intermitotic time could be calculated Control animals were subjected to exactly the (5) from the following formula: same handling procedures and confinement as the Mitotic time irradiated animals. The mice were irradiated in a Intermitotic time Mitotic index ' flat Incite cage and allowed free movement during exposure. The radiation was delivered by a 2.50-kv. peak voltage x-ray machine with no filters. The It has been shown that ionizing radiation causes inherent filtration of the x-ray tube was equiva a delay in cell division at early prophase (8, 4). At moderate dosage levels, those cells which are past lent to about 2 mm. of aluminum. The dosage de this critical stage continue through mitosis with livered to each group of mice was determined by means of a 300-r Victoreen ionisation chamber * This document is based on work performed under Con placed at the center of the cage. tract No. 7405-eng-36 for the Atomic Energy Commission. To study the effect of x-rays on the bone mar t With the technical assistance of Eletti Herring, Clare row, eight groups of seven mice were carried Morrison, Joan Thrap, and Julie Wellnitz. through the following procedure. Five animals ÃŽThe authors wish to acknowledge the assistance and advice of Gerold Tenney and his staff in the development of ir were irradiated with 400 r of x-ray given at the radiation technics and dosage measurements. rate of 100 r per minute. Animals were killed by Received for publication, September 9, 1949. crushing the cervical spine at (i, 12, 18, 24, and 30 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1950 American Association for Cancer Research. Cancer Research minutes after exposure. The control animals were calculate the normal mitotic index. The number of killed before and after the irradiated mice. A speci mitoses per 100 crypts of Lieberkühnand the aver men of hone marrow was removed from the femur age number of cells per crypt were determined for by inserting a needle into the medullary cavity and the jejunum. In the adrenal gland, the nun ber of forcing out the marrow. The specimen was mixed mitoses found in the zonae glomerulosae and fas- with a drop of fresh blood serum (human or rat) ciculatae was enumerated for 15 sections. Repre on a clean glass slide and a bone marrow smear sentative sections were then projected on white prepared. This was then air-dried, stained with paper, and the outer border of the zona glomeru- Wright's stain, and mounted in Canada balsam. losa and the inner border of the zona fasciculata The preparations were read to determine the num were outlined. The area of these two portions of ber of mitoses per 5,000 nucleated cells. Each the gland was determined by the use of a planim- mitosis was classified according to its stage of di eter. After counting the number of cells in the vision and its series, whether erythrocytic or zonae glomerulosae and fasciculatae of four of the myelocytic. Sample counts were performed on adrenal glands which had been projected, it was each bone marrow preparation to determine the possible to determine the number of cells per unit relative number of white blood cells and nucleated area of the projection. The number of mitoses per red blood cells, so that a normal mitotic index unit area and the normal mitotic index were then could be calculated for these two kinds of cells. calculated. The mitotic index of the ovarian fol licles was determined by counting the number of TABI.K1 mitoses per 50 follicles with diameters of 50 n or BONKMARROW more and then finding the average number of CHANGEIN MITOTIC ACTIVITYAFTER 400 r OF X-HAY cells per follicle. MITOSES PEH 5,000 NTCLEATED("KLLH In an effort to standardize the technic of count Time after Nucleated red ing mitotic figures, a cell was considered to be in exposure Myelocyte» blood cells mitosis only between the time, in prophase, of control 11.2+1.1 8.4+1.1 (i min. 8.0 + 1.7 54 + 10 elongation of the chromosomes before the break 1«min. 8.4 + 1.1 4.1+0.7 down of the nuclear membrane; and the time, in 18 min. 6.8±1.2 8 6±0 8 telophase, of complete separation of the daughter ¿4min. 3.9 + 0.9 s.a+o.ß 30 min. 2.7±0.9 a.o+o.s cells. This definition of mitosis was very satisfac , -V/Z (* - i tory for all tissues except the epidermis, in which Standard error \n(n-l the nuclear structure was distorted slightly by treatment with acid; therefore, the time of break To determine the mitotic time of various other down of the nuclear membrane was considered the mouse tissues, 72 mice were divided into three beginning of mitosis. groups of 24 animals each. These groups were giv en 0, 50, and 200 r of x-ray, respectively, at a rate RESULTS of 50 r per minute. Eight animals from each group There was a fairly uniform decrease in the mi were sacrificed at 10, 20, and 30 minutes after the totic activity of the myelocytes and nucleated red midpoint of exposure, or, in the case of control ani blood cells after irradiation (Table 1). mals, the midpoint of confinement. The tissues to A straight line, calculated to fit the experimen be studied were removed in a rapid manner (11) tal points for the mitotic activity in the myelo and in a definite order (6). The ears were removed cytes (Fig. 1), intersects the time ordinate at about first and placed in 0.5 per cent acetic acid; then the 35 minutes. In the case of the nucleated red blood right inguinal lymph node, a segment of jejunum, cells (Fig. 2) there is a flattening of the curve at the the right adrenal gland, and the right ovary were 24- and 30-minute points. If these points are neg removed and placed in Bouin's fixative. The epi lected, and a straight line is calculated to fit the dermis was separated from the inner surface of the 0-, 6-, 12-, and 18-minute points, this line inter ears, after 24 hours in acetic acid at a temperature sects the time ordinate at about 29 minutes. of 8°C.,and (¡-/xsectionswere prepared from the The results obtained in the other five tissues are other tissues after routine histological procedures. shown in Table 2. The responses seen in the jeju The mitotic activity of each tissue was then de num, ovary, and lymph node are almost identical termined by the methods previously described (6). (Figs. 3, 4, and 5). If the slope between the 20- and The number of mitoses per 100 standard fields, as 30-minute points for the 200-r dosage is used to outlined by a Whipple disc, and the number of determine the mitotic times of these three tissues, cells per field were determined for the epidermis they are found to be 24, 21, and 23 minutes, re and the lymph nodes, thereby making it possible to spectively.